Erik J. Sontheimer

Precision engineering of CRISPR/Cas9 components has advanced genome editing toward therapeutic applications. Completely chemically stabilized guide RNAs (gRNAs) have the potential to improve in vivo editing efficacy while enabling greater flexibility in delivery strategies. However, previous generations of fully modified guides have been associated with reduced Cas9 activity. Here, we employed an iterative, structure-guided optimization strategy to systematically introduce chemical modifications at each position of SpyCas9 gRNAs. Extending beyond commonly used nucleotide modifications, we incorporated 2’-amino-RNA, 4’-thio-RNA, and extended nucleic acid (exNA) to generate gRNA designs in which 90-100% of the nucleotides are sugar- or backbone-modified. Although certain modification patterns exhibit sequence-dependent variability, we have established a growing repertoire of guides that consistently maintain or enhance editing efficacy when applied both in vitro and in vivo . Collectively, our heavily and fully modified gRNAs hold potential for applications in nuclease editing, base editing, and other genome editing tools. Graphical

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy^1,2. Twin prime-editing guide RNAs have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs^3-6. Here we describe a prime assembly (PA) approach for the insertion of large DNA donor fragments, of which the ends are designed to overlap with the flaps generated by twin prime editing (twinPE). We used PA to insert one or multiple overlapping DNA fragments, with total insertion sizes ranging from 0.1 kb to 11 kb. An inhibitor of non-homologous end joining enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced, does not require co-delivery of exogenous DNA-dependent DNA polymerases and proceeds in non-cycling cells, suggesting independence from canonical homology-directed repair pathways. Our study demonstrates that PA can initiate Gibson-like assembly in cells to generate gene insertions without double-stranded DNA breaks, recombinases or homology-directed repair.

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy ^1,2 . Twin prime editing (PE) guide RNAs (pegRNAs) have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs ^3–6 . Here we describe a Prime Assembly (PA) approach for the insertion of large DNA donor fragments, whose ends are designed to overlap with the flaps generated by twinPE. We used PA to insert one, two, or three overlapping DNA fragments, with total insertion sizes ranging from 0.1 to 11 kilobase pairs. An inhibitor of non-homologous end joining (NHEJ) enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced and does not require co-delivery of exogenous DNA-dependent DNA polymerases. Our study demonstrates that PA can initiate “Gibson-like” assembly in cells to generate gene insertions without double-stranded DNA breaks or recombinases.

MicroRNAs (miRNAs) are small regulatory RNAs that destabilize partially complementary transcripts and cleave perfectly paired targets. miRNAs are often expressed in a specific tissue, allowing miRNA-directed cleavage to be used to prevent genome editing or gene replacement therapy in unintended cell types, a strategy called detargeting. miRNA intracellular concentration influences the potency of gene silencing, yet the absolute steady-state levels of just a few miRNAs have been determined in mammalian tissues. Here, we report the absolute abundance of miRNAs in 14 human and mouse cell lines and 17 mouse tissues, including eight brain regions. Our experiments in human cultured cells demonstrate that both miRNA and target levels influence efficacy of cleavage of fully complementary transcripts. We report the miRNA concentration required for productive cleavage of highly expressed transcripts and identify mouse miRNAs that reach this threshold in vivo.

Alpha-1 antitrypsin deficiency (AATD) is commonly caused by a G-to-A mutation in the SERPINA1 gene (the PiZ mutation). The mutant PiZ AAT protein is sequestered in hepatocytes, causing lung emphysema due to insufficient AAT protein to inhibit neutrophil elastase in the lung. Here we show that a compact adenine base editor (ABE) with an evolved Cas9 nickase derived from Neisseria meningitidis (eNme2.C) can be packaged in a single AAV and correct the PiZ mutation in mouse models of AATD. An all-in-one eNme2.C-TadA8e/guide 2 plasmid achieved approximately 20% on-target editing in PiZ reporter cells. TadA9e, which has a narrower editing window than TadA8e, reduced bystander editing without significantly affecting the on-target edit. In PiZ transgenic mice, eNme2.C-TadA9e AAV showed approximately 23% editing efficiency after 8 weeks and reduced liver disease burden in treated mice. In a new AAT-null;PiZ transgenic mouse model, ABE restored serum levels of AAT to beyond the 570 ug/mL therapeutic level. Moreover, ABE treatment was able to significantly correct lung functions in AAT-null;PiZ animals with emphysema. This study demonstrates the feasibility of an eNme2.C-based ABE in a single AAV to treat both AATD-associated liver and lung disease.

Reverse transcriptase (RT) has been shown to play a role in double-strand break repair in bacteria, yet the impact of the RT component of prime editors (PEs) on normal mammalian cellular functions is unclear. Here we show that overexpressed RT or PE increases short insertions and diminishes homology-directed repair following Cas9 cleavage at multiple loci in multiple cell lines. Live-cell imaging shows that RT and PEs are rapidly recruited to laser-induced DNA damage sites and promote endogenous repair, independent of known DNA damage sensors. Interestingly, RT–mCherry partially impairs green fluorescent protein–PARP1 recruitment. A compact PE without an RNase H domain shows reduced DNA repair activity and may therefore be more suitable for clinical application. These data reveal a role for untethered RT or the RT domain of PEs in the repair of chromosomal breaks, calling for evaluation of the long-term effect of PEs and retroviral RT in mammalian cells.

CRISPR-Cas genome editing tools enable precise, RNA-guided modification of genomes within living cells. The most clinically advanced genome editors are Cas9 nucleases, but many nuclease technologies provide only limited control over genome editing outcomes. Adenine base editors (ABEs) and cytosine base editors (CBEs) enable precise and efficient nucleotide conversions of A:T-to-G:C and C:G-to-T:A base pairs, respectively. Therapeutic use of base editors (BEs) provides an avenue to correct approximately 30% of human pathogenic variants. Nonetheless, factors such as protospacer adjacent motif (PAM) availability, accuracy, product purity, and delivery limit the full therapeutic potential of BEs. We previously developed Nme2Cas9 and its BE derivatives, including ABEs compatible with single adeno-associated virus (AAV) vector delivery, in part to enable editing near N ₄ CC PAMs. Further engineering yielded domain-inlaid BEs with enhanced activity, as well as Nme2Cas9/SmuCas9 chimeras that target single-cytidine (N ₄ C) PAMs. Here we further enhance Nme2Cas9 and Nme2 ^Smu Cas9 editing effectors for improved efficiency and vector compatibility through site-directed mutagenesis and deaminase linker optimization. Finally, we define the editing and specificity profiles of the resulting variants by using paired guide-target libraries.

In primary cell types, intracellular deoxynucleotide triphosphate (dNTP) levels are tightly regulated in a cell cycle-dependent manner. We report that prime editing efficiency is increased by mutations that improve the enzymatic properties of Moloney murine leukemia virus reverse transcriptase and treatments that increase intracellular dNTP levels. In combination, these modifications produce substantial increases in precise editing rates.